Figure 3. NR2E3 interacts with CRX.

A) HEK293T cells were transiently transfected with 3 µg of RLuc, RLuc/GFP²-NR2E3, GFP²-NR2E3/Crx-RLuc and Crx-RLuc/GFP² vectors. BRET² analysis was performed 48 h after transfection. First we quantified the GFP² fluorescence (at 515 nm) after an excitation at 485 nm (upper graph in black). Then, we added the renilla luciferase substrate, Coelanterazine h, to the cell and measured the luciferase luminescence (middle graph in white). Finally, BRET² ratio was measured in the four different conditions (lower graph in grey). B) HEK293T cells were transiently transfected with 3 µg of NR2E3-GFP², GFP²-NR2E3, Crx-RLuc and RLuc-Crx vectors, as indicated. Control transfections were performed with renilla alone (RLuc) and NR2E3 fused to GFP² (NR2E3-GFP² and GFP²-NR2E3). BRET² results (grey column) and GFP² fluorescence (black column) were expressed as mean±SEM of 2 experiments. *p<0.0001 vs. Ctrl. C) We transfected HEK293T cells with 3 µg of RLuc or Crx-RLuc vectors to keep the expression level of both proteins stable in each condition. At the same time, we transfected cells with increasing concentrations (0.5 µg to 2 µg) of NR2E3-GFP² vector. BRET² ratio was measured and sesults were expressed as mean±SEM of 3 experiments, *p<0.0001 vs. Ctrl (see also supplemental figure 1B).